Fatty acids, including polyunsaturated fatty acids (abbreviated as PUFA), are particularly important biological compounds because they intervene in many biological processes such as the construction and the maintaining of cell membranes, the synthesising of hormones (for example prostaglandins) that play a role in platelet aggregation, inflammation processes and the immunological response, etc.
Most PUFAs can be synthesised by the human body, except for two families of PUFAs which have to be taken through food, called essential fatty acids.
The two families of essential fatty acids are:                omega-6, which are particularly abundant in the oils of walnut, sunflower, soy, grape seeds or corn and fatty poultry (such as duck);        omega-3 which are especially present in the oils of walnut, in plants such as colza and flaxseed and in fatty fish (such as salmon, tuna, sardine, mackerel or herring). Methods for producing omega-3 using cultures of micro-algae, transgenic yeasts or krill have been developed recently.        
Omega-3 are PUFAs that are particularly interesting for their antioxidant virtues. Among these omega-3, EPA (eicosapentaenoic acid, C20-5ω3) and DHA (docosahexaenoic acid, C22-6ω3) purified and their enriched combinations thereof are the most commonly used as dietary supplements or as drugs in order to reduce the triglyceride levels, cardiovascular risks, improve cognition or vision, etc.
Recent clinical studies have shown that treating patients who have a triglyceride level above 500 ml/dL with 4 grams a day of ethylic ester of EPA at 96% without DHA would make it possible to reduce the triglyceride level, without increasing the LDL level (“bad” cholesterol), while treating with 4 grams a day of a mixture of ethylic esters of EPA and of DHA, at about 50% and 35% respectively, led to an increase in the LDL level concomitant with the decrease in triglycerides.
Until now, the dietary supplements of PUFA used, in particular omega-3, are substantially based on mixtures containing 30 to 60% of a mixture of EPA and DHA. In the separation methods used today, the mixture is obtained via transesterification of the triglycerides into ethylic esters then by an enrichment of the omega-3 via molecular distillation and/or co-crystallisation of saturated and mono-unsaturated fatty acids with urea. The enriched ethylic esters are possibly reconverted into triglycerides chemically or preferably enzymatically.
However, these methods of separation are not satisfactory for the production of an omega-3 such as EPA, DHA or stearidonic acid (SDA, C18-Sω3) at more than 80%, and even at more than 96%, in particular in esterified form.
However, the purification of omega-3 is delicate as these compounds include several double carbon-carbon bonds that make them sensitive to oxidation or degradation. In the presence of oxygen and when they are heated, these PUFAs undergo in particular reactions of isomerisation, oxidation, peroxidation and oligomerisation.
As such, the separation techniques mentioned hereinabove make it possible to obtain a mixture of PUFA with a good output and an acceptable degree of purity; but they cannot be implemented for the individual separation of the PUFAs. They therefore do not make it possible to separate omega-3 between them. Indeed, the molecular distillation, for example, cannot economically eliminate the DHA from EPA or SDA; it does not allow for an effective separation of the long-chain omega-3 of the C20 and C22 type. The combination of clathration with urea and the molecular distillation make it possible to obtain omega-3 mixtures with higher purity, at the price of a generally lower output and a high operating cost, but cannot be used for separating long-chain omega-3 between them, and from EPA and DHA in particular.
There is therefore a need to provide a method for the industrial purification of omega-3 in esterified form with very high purity.
Chromatography is a fine separation technique that allows for the effective purification or the enrichment of molecules in gentle conditions away from light and air.
This technology is based on the separation of molecules which are placed into contact with a stationary phase with which they have different interactions. The use of one or several fluids, referred to as mobile phases or eluents, allows for the percolation of the various molecules at different speeds. These different speeds make it possible to physically separate the molecules and to collect them in purified form at the end of chromatographic methods with one or several columns. The purified fractions are in general concentrated, in gentle conditions at ambient or moderate temperature, by means such as evaporation in a vacuum or membrane methods.
In certain cases, the starting product of the chromatographic purification is an oil comprised of fatty acid esters already enriched through molecular distillation, comprising preferably more than 30% of omega-3 of interest, which has been subjected to a treatment for eliminating oxidised compounds, or by the last molecular distillation, or through adsorption, preferably on silica derivatives (silica gel, bentonite, diatomaceous earth) or on active charcoal for example.
A certain number of chromatographic methods have been described, for obtaining omega-3 with a high purity.
As such, document U.S. Pat. No. 5,719,302 describes a method wherein the PUFAs are separated in particular using a supercritical eluent (carbonic gas under pressure), and in particular on SMB (“Simulated Moving Bed”).
Document US 2011/0091947 describes another method for purifying omega-3 that uses the technique of simulated moving bed chromatography. The document in particular describes the succession of a step of enzymatic transesterification, of two steps of molecular distillation, and of a step of the SMB type, these last three steps make it possible to separate the products into two fractions by order of retention time.
Document WO 2011/080503 describes the purification of omega-3 using a device comprising two SMB chromatographic devices arranged in series and a washing zone, with each SMB chromatographic device defining a separation zone and being comprised of several columns. The load to be treated is injected into a first separation zone in order to obtain a flow of extract and a flow of raffinate, said flow of raffinate comprising the compounds of interest then being injected into a column of the second separation zone that is not adjacent to a column of the first zone.
Document WO 2013/005051 describes the purification of omega-3 by two chromatographic separations by SMB or AMB (Actual Moving Bed) in reversed phase with a hydro-organic eluent, wherein the two separations by SMB or AMB are carried out sequentially on the same chromatographic device, or on two different devices, with the intermediary purified by the first device being introduced into the second.
Document WO 2013/005048 describes the purification of EPA at more than 90% of purity by a first chromatographic separation followed by two chromatographic separations by SMB or AMB in reversed phase with a hydro-organic eluent at each step, with the intermediary purified by the first chromatographic separation being introduced into the second chromatographic separation, and with the intermediary purified by the second chromatographic separation being introduced into the third chromatographic separation.
There is again a need to provide a method for purifying fatty acid, preferably polyunsaturated, that can be implemented in a chromatographic installation that is simpler than those of prior art, with moreover a high specific productivity (mass of purified product by mass of stationary phase and by unit of time) and a low consumption of solvents, in such a way as to reduce investment costs.